Synthesis of 3alpha-hydroxy-21-(1'-imidazolyl)-3beta-methoxyl-methyl-5alpha-pregnan-20-one via lithium imidazole with 17alpha-acetylbromopregnanone

The synthesis of biologically active 3alpha-hydroxyl-21-(1'-imidazolyl)-3beta-methoxymethyl-5alpha-pregnan-20-one was accomplished in six steps. The key steps were the improvement of stereoselectivity for acetyl isomers in C-17 and the introduction of imidazole into the core structure by use of lithium imidazole. This latter key step provided the desired product in 82% yield without the formation of 1,3-disubstituted imidazolium salt as impurity, which is generally observed in traditional method.     

Serum zinc is decreased in Alzheimer’s disease and serum arsenic correlates positively with cognitive ability

Zinc, copper, and iron aggregate Aβ and accumulate in Alzheimer’s disease (AD) plaques. Some metals are increased in AD vs. control serum. The authors examined levels of 12 metals in serum of 44 AD and 41 control subjects. Zinc decreased from 12.3 to 10.9 μmol/L (means, p = 0.0007). Arsenic positively correlated with Mini-Mental State Examination score (p < 0.0001). Zinc deposition in brain amyloid might deplete zinc from other body compartments, such as serum. The arsenic correlation might be caused by the major contribution of seafood consumption to intake of both arsenic and docosahexaenoic acid, of which the latter may delay AD.     

The Solution Structure of Heparan Sulfate Differs from That of Heparin: IMPLICATIONS FOR FUNCTION*

The highly sulfated polysaccharides heparin and heparan sulfate (HS) play key roles in the regulation of physiological and pathophysiological processes. Despite its importance, no molecular structures of free HS have been reported up to now. By combining analytical ultracentrifugation, small angle x-ray scattering, and constrained scattering modeling recently used for heparin, we have analyzed the solution structures for eight purified HS fragments dp6–dp24 corresponding to the predominantly unsulfated GlcA-GlcNAc domains of heparan sulfate. Unlike heparin, the sedimentation coefficient s_20,w of HS dp6–dp24 showed a small rotor speed dependence, where similar s_20,w values of 0.82–1.26 S (absorbance optics) and 1.05–1.34 S (interference optics) were determined. The corresponding x-ray scattering measurements of HS dp6–dp24 gave radii of gyration R_G values from 1.03 to 2.82 nm, cross-sectional radii of gyration R_XS values from 0.31 to 0.65 nm, and maximum lengths L from 3.0 to 10.0 nm. These data showed that HS has a longer and more bent structure than heparin. Constrained scattering modeling starting from 5,000 to 12,000 conformationally randomized HS structures gave best fit dp6–dp24 molecular structures that were longer and more bent than their equivalents in heparin. Alternative fits were obtained for HS dp18 and dp24, indicating their higher bending and flexibility. We conclude that HS displays bent conformations that are significantly distinct from that for heparin. The difference is attributed to the different predominant monosaccharide sequence and reduced sulfation of HS, indicating that HS may interact differently with proteins compared with heparin.     

The functional spectrum of low-frequency coding variation

Background

Rare coding variants constitute an important class of human genetic variation, but are underrepresented in current databases that are based on small population samples. Recent studies show that variants altering amino acid sequence and protein function are enriched at low variant allele frequency, 2 to 5%, but because of insufficient sample size it is not clear if the same trend holds for rare variants below 1% allele frequency.

Results

The 1000 Genomes Exon Pilot Project has collected deep-coverage exon-capture data in roughly 1,000 human genes, for nearly 700 samples. Although medical whole-exome projects are currently afoot, this is still the deepest reported sampling of a large number of human genes with next-generation technologies. According to the goals of the 1000 Genomes Project, we created effective informatics pipelines to process and analyze the data, and discovered 12,758 exonic SNPs, 70% of them novel, and 74% below 1% allele frequency in the seven population samples we examined. Our analysis confirms that coding variants below 1% allele frequency show increased population-specificity and are enriched for functional variants.

Conclusions

This study represents a large step toward detecting and interpreting low frequency coding variation, clearly lays out technical steps for effective analysis of DNA capture data, and articulates functional and population properties of this important class of genetic variation.     

Viroporin Activity of the Foot-and-Mouth Disease Virus Non-Structural 2B Protein

Viroporins are a family of low-molecular-weight hydrophobic transmembrane proteins that are encoded by various animal viruses. Viroporins form transmembrane pores in host cells via oligomerization, thereby destroying cellular homeostasis and inducing cytopathy for virus replication and virion release. Among the Picornaviridae family of viruses, the 2B protein encoded by enteroviruses is well understood, whereas the viroporin activity of the 2B protein encoded by the foot-and-mouth disease virus (FMDV) has not yet been described. An analysis of the FMDV 2B protein domains by computer-aided programs conducted in this study revealed that this protein may contain two transmembrane regions. Further biochemical, biophysical and functional studies revealed that the protein possesses a number of features typical of a viroporin when it is overexpressed in bacterial and mammalian cells as well as in FMDV-infected cells. The protein was found to be mainly localized in the endoplasmic reticulum (ER), with both the N- and C-terminal domains stretched into the cytosol. It exhibited cytotoxicity in Escherichia coli, which attenuated 2B protein expression. The release of virions from cells infected with FMDV was inhibited by amantadine, a viroporin inhibitor. The 2B protein monomers interacted with each other to form both intracellular and extracellular oligomers. The Ca²⁺ concentration in the cells increased, and the integrity of the cytoplasmic membrane was disrupted in cells that expressed the 2B protein. Moreover, the 2B protein induced intense autophagy in host cells. All of the results of this study demonstrate that the FMDV 2B protein has properties that are also found in other viroporins and may be involved in the infection mechanism of FMDV.     

Effects of Guanyl Nucleotides on [3H]Flunitrazepam Binding to Rat Hippocampal Synaptic Membranes: Equilibrium Binding and Dissociation Kinetics

The effects of guanyl nucleotides on the binding of [3H]flunitrazepam to rat hippocampal synaptic membranes were studied. In equilibrium binding studies, gamma-amino-n-butyric acid (GABA) increased and GTP decreased the binding affinity of [3H]flunitrazepam; GTP also caused a decrease in binding capacity. The effect, however, is variable. In studies of the dissociation kinetics of [3H]flunitrazepam using diazepam and the antagonist Ro 15-1788 as the displacers, there was evidence of two dissociation rate constants. GTP increased both the fast- and slow-dissociation rate constants and increased the ratio of the slow-dissociation binding state. The effect of GTP was mimicked by its nonhydrolyzable analogue 5'-guanylylimidodiphosphate but not by ATP and occurred when diazepam, but not when Ro 15-1788, was used as the displacer. GABA antagonized the effect of GTP on the dissociation of [3H]flunitrazepam. The nature of the benzodiazepine receptor, its actions, and the possible role of cyclic AMP as a second messenger are discussed.     

Microtubule-associated protein light chain 2 is a stargazin-AMPA receptor complex-interacting protein in vivo

The ataxic mutant mouse stargazer is a null mutant for stargazin, a protein involved in the regulation of cell surface trafficking and synaptic targeting of AMPA receptors. The extreme C terminus of stargazin (sequence, -TTPV), confers high affinity for PDZ domain-containing proteins e.g. PSD-95. Interaction with PDZ proteins enables stargazin to fulfill its role as an AMPA receptor synaptic targeting molecule but is not essential for its ability to influence AMPA receptor trafficking to the neuronal cell surface. Using the yeast-two hybrid approach we screened for proteins that interact with the intracellular C-terminal tail of stargazin. Positive interactors included PDZ domain-containing proteins e.g. SAP97, SAP102, and PIST. Interestingly, light chain 2 of microtubule-associated protein 1 (LC2), which does not contain a PDZ domain, was also a strong interactor. This was shown to be a direct interaction that occurred upstream of the -TTPV sequence of stargazin. Immunoprecipitations of Triton X-100 soluble cerebellar extracts revealed that LC2 is pulled down not only by anti-stargazin antibodies but also anti-GluR2 antibodies suggesting that stargazin and AMPA receptor subunits associate with LC2. Immunopurified full-length, native stargazin was shown to co-associate not only with GluR2 in vivo but also with full-length, native LC2. Indeed, LC2 co-associates with stargazin when part of a tripartite complex comprising LC2-stargazin-GluR2. Since this complex was extracted using Triton X-100 and was devoid of PSD95, SAP97, and actin we postulate that LC2 is involved in trafficking of AMPA receptors in cerebellar neurons before they are anchored at the synapse.